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,
,,§
*
Virginia Mason Research Center, Seattle, WA 98101;
Fred Hutchinson Cancer Research Center, Seattle, WA 98104; and Departments of
Immunology and
§
Medicine, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-2 binds to a cell surface receptor complex composed of three
distinct chains, IL-2R
, IL-2Rß, and 
c (10), inducing catalytic
activation of the Janus kinases Jak1 and Jak3 (11, 12, 13). Activation of
these tyrosine kinases requires the presence of a serine-rich, or
S,3 region within the
membrane-proximal 86 cytoplasmic residues of IL-2Rß (14). These 86
amino acids contain Box1 and Box2 motifs found in many members of the
hemopoietic receptor superfamily (15). Although Jak1 appears to be
dispensable for IL-2-mediated mitogenesis (16), Jak3 activation is
essential, since 1) IL-2 fails to mediate a proliferative signal in
Jak3-deficient mice (17, 18), and 2) overexpression of a catalytically
inactive version of Jak3 in the pro-B cell line BA/F3 inhibits the
induction of c-myc and subsequent cell proliferation
promoted by IL-2 (19).
In addition to catalytic activation of Jak3, tyrosine phosphorylation
of IL-2Rß is necessary for proliferation, since point mutation of
three specific cytoplasmic tyrosine residues of IL-2Rß to
phenylalanine abrogates both phosphorylation of this receptor subunit
and mitogenic signaling (20). By contrast, the cytoplasmic tyrosine
residues of c are dispensable for mitogenic signaling (21, 22).
IL-2Rß contains six cytoplasmic tyrosines (Y338, Y355, Y358, Y361,
Y392, and Y510), all of which are distal to the S region. Y338, Y355,
Y358, and Y361 are located in a segment referred to as the acidic or A
region, whereas Y392 and Y510 are in the C-terminal H region (Fig. 1
).
Although Y355, Y358, and Y361 are completely dispensable for
mitogenesis, the presence of at least one of the other three tyrosine
residues on IL-2Rß (Y338, Y392, or Y510) is necessary for tyrosine
phosphorylation of this chain and the generation of a proliferative
signal (20).
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In addition to promoting proliferation, the IL-2R prevents the apoptosis of activated T cell (3, 4), an event that, unlike proliferation, has not previously been specifically linked to IL-2Rß cytoplasmic tyrosines. Although it is possible that cell viability is simply a default consequence of proper cell cycle progression, there is also evidence that cytokine receptors such as the IL-2R generate antiapoptotic signals that are distinct from proliferative signals. Indeed, resting T cells can receive a nonmitogenic survival signal from IL-2 that, unlike the IL-2 proliferative signal, is not dependent upon Jak3 activation, and instead requires activation of the lipid kinase phosphatidylinositol-3 kinase (36). Moreover, the transcription factor STAT3 appears to mediate a signal for cell survival, but not proliferation, from the IL-6R chain gp130 by inducing the proto-oncogene bcl-2 (32). bcl-2 and the related gene bcl-x, both of which are induced by IL-2 (2, 9, 28), have been proposed to promote cell viability because constitutive overexpression of either gene in cytokine-dependent cell lines can significantly delay the onset of cytokine starvation-mediated apoptosis in the absence of proliferation (6, 7, 9) However, it is not known whether physiologic expression of these genes is sufficient to account for the ability of cytokines to prevent apoptosis.
In this study, we have analyzed the contribution of Shc to proliferation and survival signals mediated by the IL-2R. We demonstrate that covalently linking Shc to a truncated version of IL-2Rß lacking all cytoplasmic tyrosines restores the ability of the receptor to promote c-myc induction and cell proliferation, thereby directly demonstrating a role for Shc in IL-2-mediated mitogenesis. Additionally, the IL-2Rß/Shc fusion protein mediates induction of the antiapoptotic genes bcl-2 and bcl-x with normal kinetics. Nonetheless, this Shc-mediated signal is insufficient to maintain cell survival for more than a few days. Thus, IL-2R-induced proliferation and bcl-family gene expression are not sufficient to maintain the long-term survival of activated T cells. Rather, Y338 of IL-2Rß appears to activate an unknown pathway independent of Shc that leads to downstream events that are necessary for long-term cell viability.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression vectors encoding the chimeric - and
ßß-chains (formerly denoted GM/2 and GMß/2ß) under the
control of the human ß-actin promoter have been described previously
(37). The signal peptide of ßß was replaced with that of the
GM-CSFR-chain to improve expression of ßß (B.H.N., unpublished
results). Truncations of ßß were generated by PCR with
oligonucleotides encoding premature stop codons, and PCR products were
cloned between a unique AflII site in the cytoplasmic domain
of ßß and a unique XbaI site immediately 3' to the stop
codon of ßß. Point mutations were generated by the splice-overlap
extension PCR technique (38), and the PCR products were similarly
inserted between the unique AflII and XbaI sites.
To generate ßß325-Shc, the murine Shc cDNA was first crudely
ligated C-terminal to ßß. A specific oligonucleotide encoding an
AflII site separated from the N-terminal methionine of p52
Shc by four glycine residues was then used to synthesize, by PCR, an
in-frame fusion between Ser324 of ßß and the N-terminal
methionine of Shc. This PCR product was ligated between the unique
AflII site of ßß and a unique BamHI site in
Shc. All regions of IL-2Rß generated by PCR were sequenced with the
Applied Biosystems Prism dye terminator cycle sequencing kit
(Perkin-Elmer, Norwalk, CT).
Cell culture
The murine IL-2-dependent T cell line CTLL-2 was obtained from
American Type Culture Collection (Manassas, VA) and maintained in 10%
FCS, 45% RPMI (Life Technologies, Gaithersburg, MD), 45% Click’s
media (Altick Enterprises, River Falls, WI), supplemented with 2 mM
L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 1
mM sodium pyruvate, 0.07% sodium bicarbonate, 5 mM HEPES, 25 mM
ß-mercaptoethanol, and, unless otherwise specified, 50 U/ml human
rIL-2 (Chiron, Emeryville, CA). Linearized plasmids were introduced
into cells by electroporation, and stable transfectants were selected
for resistance to G418 in 96-well plates at limiting dilution to
isolate independent subclones. Receptor expression was assessed by flow
cytometry with Abs to human GM-CSFR or ßc (Santa Cruz
Biotechnology, Santa Cruz, CA). Subclones with comparable receptor
expression were chosen for further analyses.
Proliferative assays
Thymidine incorporation assays were conducted in triplicate
wells with 104 cells/well exposed to titrated doses of
GM-CSF or IL-2 for 20 h, followed by a 4-h
[3H]thymidine pulse (2.5 µCi/well). Cells were
harvested onto glass fiber filters, and DNA synthesis was quantitated
by liquid scintillation counting. The data presented in Figures 1
and 3
were based on a dose of GM-CSF (100 ng/ml) that was found to elicit a
maximal response from all functional receptor mutants.
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Cells that had been stimulated as indicated were washed once with PBS and then lysed on ice in lysis buffer (0.05 M, pH 7.4, Tris base, 0.5% Nonidet P-40, 0.15 M sodium chloride, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF or AEBSF-4-(2-aminoethyl)benzene sulfonyl fluoride, 10 mg/ml leupeptin, and 10 mg/ml aprotinin). Nuclei were removed by centrifugation, and cytoplasmic fractions were precleared with rabbit anti-rat Ig antiserum (Boehringer Mannheim, Indianapolis, IN) and protein A-agarose (Santa Cruz Biotechnology). Shc protein was then immunoprecipitated with polyclonal rabbit antiserum (Transduction Laboratories, Lexington, KY) and protein A-agarose (Santa Cruz Biotechnology). Agarose-bound immune complexes were washed twice with lysis buffer and then boiled for 3 min in SDS sample buffer.
Western blots
Immunoprecipitated proteins were run on acrylamide gels and transferred to nitrocellulose. Nitrocellulose blots were blocked with TTBS (0.1 M, pH 7.5, Tris base, 0.9% sodium chloride, 0.05% Tween 20) containing 5% powdered skim milk (Carnation, Glendale, CA) or 1% BSA (for antiphosphotyrosine probes), and probed with murine mAbs to Shc (Transduction Labs) or phosphotyrosine (Upstate Biotechnology, Lake Placid, NY). Blots were then washed with TTBS, probed with peroxidase-conjugated goat anti-rabbit or anti-mouse Abs (Life Technologies, Grand Island, NY), and washed again with TTBS. Bound Abs were detected by enhanced chemoluminescence (Amersham, Arlington Heights, IL, or DuPont NEN, Boston, MA). ERK1/ERK2 phosphorylation was assayed per the protocol of the PhosphoPlus MAPK Antibody Kit (New England Biolabs, Beverly, MA). Blots were stripped with a 30-min, 50°C incubation in 62.6 mM Tris-HCl (pH 6.7), 0.1 M ß-mercaptoethanol, and 2% SDS.
Electrophoretic mobility shift assays
Cells that had been stimulated as indicated in the Figure 4
legend were washed once with buffer H (20 mM, pH 7.9, HEPES, 1 mM EDTA,
0.1 mM EGTA, 2 mM magnesium chloride, 1 mM sodium orthovanadate, 20 mM
sodium fluoride, 1 mM DTT, 0.1 mM AEBSF-4-(2-aminoethyl)benzene
sulfonyl fluoride, and 1 mg/ml leupeptin) and lysed in buffer H plus
0.2% Nonidet P-40 at 0°C. Nuclei were pelleted by centrifugation and
then extracted with buffer K (buffer H plus 0.42 M sodium chloride and
20% v/v glycerol). A probe for STAT activity was generated with
incompletely overlapping oligonucleotides corresponding to the sense
and antisense strands of the STAT-responsive DNA element from the
FcRI promoter, which were annealed, radiolabeled with
[-32P]dCTP by an end-filling T4 polymerase reaction,
and purified with a MicroSpin G-25 column (Pharmacia, Piscataway, NJ).
The probe was added to nuclear extracts in 50 mM potassium chloride, 10
mM HEPES (pH 7.9), 10% glycerol, 1 mM DTT, and 87.5 mg/ml dITP/dCTP at
room temperature for 30 min, and the reaction mixture was run on an
acrylamide gel, followed by autoradiography.
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Cells that had been stimulated as indicated were pelleted by
centrifugation and flash frozen in a dry ice/ethanol bath. RNA was
harvested from thawed pellets with the RNA STAT 60 kit (Tel Test,
Friendswood, TX), denatured for 10 min at 65°C in 20 mM MOPS, 5 mM
sodium acetate, 0.5 mM EDTA, 2.4 M formaldehyde, and 50% formamide,
and run on a 1.2% agarose gel containing 20 mM MOPS, 5 mM sodium
acetate, 0.5 mM EDTA, and 1.1 M formaldehyde. RNA was passively
transferred to nytran membranes with 10x SSC (1.5 M sodium chloride,
0.15 M sodium citrate, pH 7), and UV cross-linked. Blots were
prehybridized at 43°C in hybridization buffer (1 M sodium phosphate
(pH 7.1), 2 mM EDTA, 2% BSA, 10% SDS, 50% formamide, and 0.16 mg/ml
yeast tRNA or herring sperm DNA). Nucleic acid probes were generated
with a 2.2-kb EcoRI fragment of rat c-fos, a
0.4-kb PstI fragment of murine c-myc, a
0.9-kb PstI fragment of murine bcl-2, a
1-kb EcoRI fragment of murine bcl-x, and a 1.2-kb
PstI fragment of murine GAPDH cDNA, which were radiolabeled
with [-32P]dCTP using a random-primed labeling kit
(Boehringer Mannheim) and purified with Centri-Sep spin columns
(Princeton Separations, Adelphia, NJ). Probes were boiled for 10
min and added to blots in hybridization buffer. After overnight
incubation at 43°C, blots were washed two to three times with
2x SSC, 0.1% SDS, once with 0.2x SSC, 0.1% SDS at room temperature,
and one to three times with 0.2x SSC, 0.1% SDS at 55°C before
autoradiography. Blots were stripped with a 2-min immersion in boiling
water before reprobing.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To perform stucture/function analyses of IL-2Rß in a T cell line
that normally responds to IL-2 and hence expresses a functional
endogenous IL-2R, we utilized a previously described chimeric
GM-CSF/IL-2R. The chimeric receptor consists of two chains, and
ßß, containing the extracellular domains of the human GM-CSF
receptor - and ß-chains fused, respectively, to the transmembrane
and intracellular regions of c and IL-2Rß. When coexpressed,
and ßß deliver in response to human GM-CSF a signal that is
biochemically and physiologically indistinguishable from that induced
in the same cell by the wild-type IL-2R (21, 37, 39) The murine
cytotoxic T lymphocyte line CTLL-2 was cotransfected with and
either full-length ßß (ßßwt), or mutated derivatives of ßß
(Fig. 1). G418-resistant subclones of
transfectants were analyzed for receptor expression by flow cytometry
(data not shown), and those with comparable expression of both -
and the various ßß-chains were chosen for further study.
While the IL-2 proliferative signal can be delivered through three
functionally redundant intracellular tyrosines on IL-2Rß (Y338, Y392,
and Y510) (20), the adapter molecule Shc is recruited to IL-2Rß
exclusively through Y338 (24). Therefore, to focus our analysis upon
the role of Shc in IL-2-mediated proliferation in the absence of
redundant signals from Y392 or Y510, residue Y355 of ßß was
replaced with a premature stop codon to generate a mutant
(ßß
355) that lacks all intracellular tyrosine residues except
for Y338 (Fig. 1). Consistent with Y338 serving as a binding site for
Shc, the ßß355 receptor was able to induce Shc phosphorylation
in response to GM-CSF unless Y338 was point mutated to phenylalanine
(ßß355Y338
F) (Fig. 2A). This point mutation also
abrogated the ability of ßß355 to induce MAP kinase
phosphorylation (Fig. 2B) and subsequent induction of the
proto-oncogene c-fos (Fig. 2C), events that are
mediated through Shc (25, 26).
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355 generated a robust proliferative signal in CTLL-2 cells
(Fig. 3A). ßß355 also
induced expression of the proto-oncogene c-myc (Fig. 3B), an event that is critical for T cell proliferation (5).
The point mutation of Y338 to phenylalanine present in the
ßß355Y338F receptor chain, however, abrogated
both c-myc induction (Fig. 3B) and proliferation
(Fig. 3A). Nonetheless, ßß355Y338F
remained competent to interact functionally with the -chain in
response to GM-CSF, as it induced Jak3 phosphorylation in response to
GM-CSF (data not shown), consistent with previous reports that IL-2Rß
cytoplasmic tyrosines are not essential for Janus kinase activation
(22). Thus, as the sole remaining cytoplasmic IL-2Rß tyrosine, Y338
can clearly mediate c-myc induction and the proliferation of
a T cell line.
Y338 has also been shown to mediate STAT5 activation in the T cell line
HT-2 (41), although unlike Shc, STAT5 can also interact with tyrosine
residues of the H region (20, 40). Consistent with a role for Y338 in
activating STAT5, ßß355 induced STAT DNA-binding activity in
CTLL-2 cells, the vast majority of which was abrogated by point
mutation of Y338 to phenylalanine in ßß355Y338F
(Fig. 4). The ability of Y338 to mediate
both Shc phosphorylation and STAT5 activation suggests that Y338
interacts with multiple molecules, raising the distinct possibility
that Y338 delivers a mitogenic signal through molecules other than Shc.
Indeed, STAT5 itself has been implicated in the proliferative signal
delivered by other cytokine receptors (42).
To specifically test the hypothesis that Y338 delivers a mitogenic
signal through association with Shc, a fusion protein (ßß325-Shc)
was constructed to force the association of Shc with IL-2Rß in the
absence of Y338. The N terminus of Shc was covalently attached, through
a flexible tetraglycine linker, to the C terminus of a truncated
version of ßß lacking all cytoplasmic tyrosines normally necessary
for proliferation (Fig. 1). The S region of IL-2Rß was retained, as
this region is necessary for Jak3 activation (14), which in turn is
necessary for Shc-mediated signaling (19, 39). We have previously
employed a similar fusion protein strategy to reconstitute the
signaling function of a truncated c-chain by covalent attachment of
Jak3 (39). The ßß325-Shc chain itself underwent inducible tyrosine
phosphorylation (Fig. 2A), presumably at sites within the
Shc sequence, which contains the only cytoplasmic tyrosines in
ßß325-Shc. Moreover, ßß325-Shc activated the MAP kinase pathway
in response to GM-CSF, as indicated by phosphorylation of MAP kinase
(Fig. 2B) and induction of c-fos (Fig. 2C). Thus, ßß325-Shc can reconstitute Shc functions and
mediate several of the biochemical events normally associated with Y338
of IL-2Rß. ßß325-Shc also induced Jak3 phosphorylation (data not
shown). However, ßß325-Shc did not induce any STAT DNA-binding
activity beyond the trace amount induced by the
ßß355Y338F receptor chain (Fig. 4), indicating
that the STAT activation mediated through Y338 in CTLL-2 cells occurs
by a Shc-independent mechanism.
Analysis of mitogenic signaling revealed that ßß325-Shc promoted
DNA synthesis in response to GM-CSF to a similar extent as did
ßß355 (Fig. 3A). ßß325-Shc also induced
c-myc expression with the same kinetics and magnitude as did
ßß355 (Fig. 3B), thereby directly demonstrating a
biochemical pathway from Shc to this proto-oncogene. The signal from
ßß325-Shc was competent for progression through the entire cell
cycle, as evidenced by the majority of subclones increasing in number
by two- to threefold after 24 h of culture with GM-CSF (Fig. 3C). This proliferative signal was specifically dependent
upon the Shc portion of the receptor, as a truncated receptor lacking
the Shc sequence and tetraglycine linker (ßß325, Fig. 1) failed
to mediate proliferation (Fig. 3A).
Shc mediates bcl-2 and bcl-x induction, but not long-term survival
The ability of cytokines to regulate cell survival has been
ascribed to their ability to regulate expression of
bcl-family genes (2, 6, 7, 9). Although the A region of
IL-2Rß, containing Y338, is not absolutely required for IL-2-mediated
bcl-2 or bcl-x induction (28), the full-length
IL-2Rß-chain must have redundant sites through which it induces these
genes, as the ßß355 receptor induced both bcl-2 and
bcl-x through a Y338-dependent mechanism (Fig. 5). Such a requirement for either Y338 or
more distal residues of IL-2Rß was obviated by covalent attachment of
Shc, as ßß325-Shc induced normal expression of both
bcl-2 and bcl-x genes (Fig. 5). Therefore, these
antiapoptotic bcl-family genes, like c-myc, are
targets of a Shc-dependent signal.
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355 receptors mediated
equivalent bcl-family gene induction, ßß325-Shc differed
from ßß355 in that it did not support the long-term culture of
CTLL-2 cells in the presence of GM-CSF. This appears to be because the
ßß325-Shc receptor fails to prevent apoptosis, for while CTLL-2
subclones could be expanded for weeks with little cell death evident
when stimulated through the ßß355 receptor (data not shown), all
subclones stimulated through ßß325-Shc demonstrated markedly
reduced viability by 48 h relative to cells expressing
ßß355, and viable cells were rare or undetectable after 6 days
(Fig. 6). Cells expressing ßß325-Shc
and cultured with GM-CSF showed a brief survival advantage over cells
expressing the ßß355Y338F receptor, or cells
cultured with media alone. However, this survival advantage was
transient, being evident primarily in the first 24 h of culture
(Fig. 6). Thus, Shc mediates several aspects of IL-2 signaling, such as
c-myc, bcl-2, and bcl-x induction and
proliferation, but is not competent to deliver antiapoptotic signals
necessary for the long-term expansion of T cells. Indeed, by promoting
proliferation in the absence of survival, ßß325-Shc demonstrates
that the IL-2R promotes mitogenesis and prevents apoptosis by discrete
intracellular signals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A) (20) and survival (Fig. 6) of
IL-2-dependent cells. While this finding suggests that Y338 may
therefore deliver proliferation and survival signals through Shc, the
fact that Y338 can also lead to activation of an alternative molecule
such as STAT5 (41) (Fig. 4) suggests that other signaling pathways may
be involved. To specifically identify the consequences of IL-2
signaling that are mediated through Shc, a novel fusion protein
(ßß325-Shc) was designed to physically associate IL-2Rß with Shc
while precluding the association of other phosphotyrosine-binding
proteins with IL-2Rß. Through this construct, we demonstrated that
Shc could indeed induce a proliferative signal in the IL-2-dependent
murine T cell line CTLL-2 (Fig. 3, A and C). Shc
also mediated expression of the promitogenic gene c-myc and
the antiapoptotic genes bcl-2 and bcl-x (Figs. 3B and 5). However, these Shc-mediated signals were
insufficient to prevent the apoptosis of CTLL-2 cells, which occurs by
default upon removal of IL-2 (Fig. 6). Thus, this study demonstrates
that the IL-2R delivers distinct signals for cell proliferation and
survival through Y338, and that only the former is mediated through
Shc.
The strategy of covalently linking a cytoplasmic signaling molecule to
a truncated receptor has been successfully applied in the past to
determine the roles of Lck in CD4 signaling (44), Jak2 in IFN-
signaling (45), and Jak3 in IL-2 signaling (39). Such a strategy has
the inherent risk that molecules may adopt a conformation or
orientation that compromises their signaling capabilities. Therefore,
we attempted to reconstitute the normal orientation of Shc relative to
IL-2Rß by linking the N-terminal region of Shc, which normally binds
IL-2Rß (23), to the C terminus of ßß325, which is only 13
residues from the tyrosine (Y338) to which Shc normally binds (Fig. 1).
Several indices of normal Shc function, including Shc and MAP kinase
phosphorylation and c-fos induction, were fully
reconstituted with the ßß325-Shc receptor chain (Fig. 2), while the
Shc-independent ability of Y338 to promote STAT activation was not
(Fig. 4).
ßß325-Shc induced both c-myc expression and cell
proliferation (Fig. 3), indicating that Shc can mediate a mitogenic
signal from the IL-2R, similar to the role Shc has been proposed to
play in EGF receptor signaling (46). ßß325-Shc also induced
bcl-2 and bcl-x expression (Fig. 5),
demonstrating that signals from Shc can activate these antiapoptotic
genes. However, IL-2R signaling must lead to induction of the
bcl-2 gene through multiple pathways, since a dominant
negative Jak3 molecule, which abrogates Shc-mediated signals, failed to
block induction of bcl-2 (19). Moreover, a full-length form
of IL-2Rß, in which all six cytoplasmic tyrosines were mutated to
phenylalanine, induced bcl-2 expression in the absence of
the Shc binding site (41). The molecular nature of this alternative
pathway to bcl-2 remains undefined.
As an adapter molecule, Shc can interact with multiple downstream signaling molecules that potentially mediate the proliferative and gene-induction signals described in this work. For example, Shc contains an N-terminal phosphotyrosine binding domain that interacts with SH2-containing 5'-inositol phosphatase and a collagen homology domain that interacts with the adapter molecule Grb2 (27, 47, 48). The ras protein is normally activated downstream of Grb2, and constitutively active versions of ras have been shown to mediate bcl-2 and bcl-x induction and, when coexpressed with c-myc, promote proliferation (49, 50). Additionally, overexpression of a dominant negative version of Shc bearing point mutations in the collagen homology domain inhibits c-myc induction by IL-3 and EGF (46, 51). Shc also contains a C-terminal SH2 domain capable of interacting with tyrosine-phosphorylated proteins, although no such interactions have yet been described in the context of the IL-2R. Future structure/function analyses of ßß325-Shc should allow determination of the domains of Shc that mediate proliferation and induction of the c-myc, bcl-2, and bcl-x genes by IL-2.
Although ßß325-Shc induced the antiapoptotic genes bcl-2
and bcl-x (Fig. 5), it was unable to support the long-term
culture of CTLL-2 cells because it failed to prevent apoptosis (Fig. 6), indicating that the induction of bcl-2 and
bcl-x by IL-2 is not sufficient to support the long-term
viability of cells. Indeed, the ability of constitutively overexpressed
bcl-2 to prevent cytokine starvation-mediated apoptosis in
factor-dependent cells is only transient (6, 9). Similarly, since
ßß325-Shc delivered a competent proliferative signal (Fig. 3, A and C), cell cycle progression is likewise not
sufficient to prevent apoptosis. Although bcl-2 or
bcl-x induction or cell cycle progression may account for
the slight survival advantage that the ßß325-Shc receptor confers
over media alone (Fig. 6), there must be an additional, presumably
Shc-independent, mechanism by which the wild-type IL-2R promotes
long-term survival. This could involve the regulation of genes not
examined in this study, for example inducing the antiapoptotic
bcl-family member A1 (52) or suppressing such
proapoptotic members as Bax or Bad (53, 54).
Alternatively, it may involve cytoplasmic events implicated in
antiapoptotic signaling, such as phosphorylation, and consequent
cytoplasmic sequestration, of the proapoptotic bcl-family
protein Bad (55) mediated by the kinase c-Akt (56, 57). However, a
defect in the latter antiapoptotic pathway is unlikely to account for
the inability of ßß325-Shc to support cell viability, as
ßß325-Shc promoted the phosphorylation of Akt on Ser473
(J.D.L., unpublished results), which is involved in the activation of
this kinase.
The ability of ßß325-Shc to induce robust proliferation (Fig. 3, A and C) without supporting cell survival (Fig. 6) indicates that the IL-2R delivers discrete signals for proliferation
and long-term survival, such as have been described in the
IL-3/IL-5/GM-CSF and IL-6 receptor systems (32, 58, 59). Because IL-2
is the principal mitogenic cytokine for mature T cells, discrete
proliferative and survival signals from the IL-2R may play a decisive
role in normal T cell physiology in vivo. For example, through the
selective attenuation of either a proliferative or survival signal from
the IL-2R, a T cell could be induced to proliferate, persist, or
apoptose upon activation, thus dictating the course of an immune
response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Brad Nelson, Virginia Mason Research Center, 1000 Seneca St., Seattle, WA 98101.
3 Abbreviations used in this paper: S region, serine-rich region; A region, acidic region; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage CSF; H region, C-terminal cytoplasmic half; IRS, insulin receptor substrate; MAP, mitogen-activated protein.
Received for publication April 27, 1998. Accepted for publication June 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression. Eur. J. Immunol. 26:294.[Medline]
)-deficient T cells expressing a transgenic antigen receptor. J. Immunol. 158:3738.[Abstract]
chains. Proc. Natl. Acad. Sci. USA 91:7271.-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction. Proc. Natl. Acad. Sci. USA 92:8724. c chain sufficient for Jak kinase activation and induction of proliferation in T cells. Mol. Cell. Biol. 16:309.[Abstract]
c chain. J. Biol. Chem. 270:21729. chains mediate the signal for T-cell proliferation. Nature 369:333.[Medline]
c chain for Janus kinase activation leading to T cell proliferation. Proc. Natl. Acad. Sci. USA 94:1878. interferon receptor in intact cells. Mol. Cell. Biol. 16:3214.[Abstract]
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 D. de Totero, R. Meazza, M. Capaia, M. Fabbi, B. Azzarone, E. Balleari, M. Gobbi, G. Cutrona, M. Ferrarini, and S. Ferrini The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways Blood, January 15, 2008; 111(2): 517 - 524. [Abstract] [Full Text] [PDF]
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 H. M. Lockyer, E. Tran, and B. H. Nelson STAT5 Is Essential for Akt/p70S6 Kinase Activity during IL-2-Induced Lymphocyte Proliferation J. Immunol., October 15, 2007; 179(8): 5301 - 5308. [Abstract] [Full Text] [PDF]
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C.-L. Hsu, K. Kikuchi, and M. Kondo Activation of mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK) signaling pathway is involved in myeloid lineage commitment Blood, September 1, 2007; 110(5): 1420 - 1428. [Abstract] [Full Text] [PDF]
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S. D. Saibil, R. G. Jones, E. K. Deenick, N. Liadis, A. R. Elford, M. G. Vainberg, H. Baerg, J. R. Woodgett, S. Gerondakis, and P. S. Ohashi CD4+ and CD8+ T Cell Survival Is Regulated Differentially by Protein Kinase C{theta}, c-Rel, and Protein Kinase B J. Immunol., March 1, 2007; 178(5): 2932 - 2939. [Abstract] [Full Text] [PDF]
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A. Yu and T. R. Malek Selective Availability of IL-2 Is a Major Determinant Controlling the Production of CD4+CD25+Foxp3+ T Regulatory Cells J. Immunol., October 15, 2006; 177(8): 5115 - 5121. [Abstract] [Full Text] [PDF]
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D. K. Taylor, P. T. Walsh, D. F. LaRosa, J. Zhang, M. A. Burchill, M. A. Farrar, and L. A. Turka Constitutive Activation of STAT5 Supersedes the Requirement for Cytokine and TCR Engagement of CD4+ T Cells in Steady-State Homeostasis J. Immunol., August 15, 2006; 177(4): 2216 - 2223. [Abstract] [Full Text] [PDF]
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S. Manicassamy, S. Gupta, Z. Huang, and Z. Sun Protein Kinase C-{theta}-Mediated Signals Enhance CD4+ T Cell Survival by Up-Regulating Bcl-xL. J. Immunol., June 1, 2006; 176(11): 6709 - 6716. [Abstract] [Full Text] [PDF]
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Y. Zhang, R. A. Kirken, L. Furian, S. Janczewska, X. Qu, W. W. Hancock, M. Wang, N. Tejpal, R. Kerman, B. D. Kahan, et al. Allograft Rejection Requires STAT5a/b-Regulated Antiapoptotic Activity in T Cells but Not B Cells J. Immunol., January 1, 2006; 176(1): 128 - 137. [Abstract] [Full Text] [PDF]
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R. Barouch-Bentov, E. E. Lemmens, J. Hu, E. M. Janssen, N. M. Droin, J. Song, S. P. Schoenberger, and A. Altman Protein Kinase C-{theta} Is an Early Survival Factor Required for Differentiation of Effector CD8+ T Cells J. Immunol., October 15, 2005; 175(8): 5126 - 5134. [Abstract] [Full Text] [PDF]
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 L. B. Baughn and N. Rosenberg Disruption of the Shc/Grb2 Complex during Abelson Virus Transformation Affects Proliferation, but Not Apoptosis J. Virol., February 15, 2005; 79(4): 2325 - 2334. [Abstract] [Full Text] [PDF]
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D. Milojkovic, S. Devereux, N. B. Westwood, G. J. Mufti, N. S. B. Thomas, and A. G. S. Buggins Antiapoptotic Microenvironment of Acute Myeloid Leukemia J. Immunol., December 1, 2004; 173(11): 6745 - 6752. [Abstract] [Full Text] [PDF]
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Y. Taguchi, T. Kondo, M. Watanabe, M. Miyaji, H. Umehara, Y. Kozutsumi, and T. Okazaki Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase Blood, November 15, 2004; 104(10): 3285 - 3293. [Abstract] [Full Text] [PDF]
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H. Zhang, D. M. Conrad, J. J. Butler, C. Zhao, J. Blay, and D. W. Hoskin Adenosine Acts through A2 Receptors to Inhibit IL-2-Induced Tyrosine Phosphorylation of STAT5 in T Lymphocytes: Role of Cyclic Adenosine 3',5'-Monophosphate and Phosphatases J. Immunol., July 15, 2004; 173(2): 932 - 944. [Abstract] [Full Text] [PDF]
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 S. Pacini, M. Pellegrini, E. Migliaccio, L. Patrussi, C. Ulivieri, A. Ventura, F. Carraro, A. Naldini, L. Lanfrancone, P. Pelicci, et al. p66SHC Promotes Apoptosis and Antagonizes Mitogenic Signaling in T Cells Mol. Cell. Biol., February 15, 2004; 24(4): 1747 - 1757. [Abstract] [Full Text] [PDF]
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J. J. Moon, E. D. Rubio, A. Martino, A. Krumm, and B. H. Nelson A Permissive Role for Phosphatidylinositol 3-Kinase in the Stat5- mediated Expression of Cyclin D2 by the Interleukin-2 Receptor J. Biol. Chem., February 13, 2004; 279(7): 5520 - 5527. [Abstract] [Full Text] [PDF]
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F. Behbod, Z. S. Nagy, S. M. Stepkowski, J. Karras, C. R. Johnson, W. D. Jarvis, and R. A. Kirken Specific Inhibition of Stat5a/b Promotes Apoptosis of IL-2-Responsive Primary and Tumor-Derived Lymphoid Cells J. Immunol., October 15, 2003; 171(8): 3919 - 3927. [Abstract] [Full Text] [PDF]
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B. H. Nelson, T. P. Martyak, L. J. Thompson, J. J. Moon, and T. Wang Uncoupling of Promitogenic and Antiapoptotic Functions of IL-2 by Smad-Dependent TGF-{beta} Signaling J. Immunol., June 1, 2003; 170(11): 5563 - 5570. [Abstract] [Full Text] [PDF]
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M. J. Lindemann, M. Benczik, and S. L. Gaffen Anti-apoptotic Signaling by the Interleukin-2 Receptor Reveals a Function for Cytoplasmic Tyrosine Residues within the Common gamma (gamma c) Receptor Subunit J. Biol. Chem., March 14, 2003; 278(12): 10239 - 10249. [Abstract] [Full Text] [PDF]
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M. Minagawa, H. Watanabe, C. Miyaji, K. Tomiyama, H. Shimura, A. Ito, M. Ito, J. Domen, I. L. Weissman, and K. Kawai Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor {beta}-Chain-Deficient Mice J. Immunol., October 15, 2002; 169(8): 4153 - 4160. [Abstract] [Full Text] [PDF]
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D. E. Isaksen, H. Baumann, B. Zhou, S. Nivollet, A. G. Farr, S. D. Levin, and S. F. Ziegler Uncoupling of Proliferation and Stat5 Activation in Thymic Stromal Lymphopoietin-Mediated Signal Transduction J. Immunol., April 1, 2002; 168(7): 3288 - 3294. [Abstract] [Full Text] [PDF]
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 L. E. Cheng, C. Ohlen, B. H. Nelson, and P. D. Greenberg Enhanced signaling through the IL-2 receptor in CD8+ T cells regulated by antigen recognition results in preferential proliferation and expansion of responding CD8+ T cells rather than promotion of cell death PNAS, February 20, 2002; (2002) 52676899. [Abstract] [Full Text] [PDF]
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S. N. Tucker, H. K. Jessup, H. Fujii, and C. B. Wilson Enforced expression of the Ikaros isoform IK5 decreases the numbers of extrathymic intraepithelial lymphocytes and natural killer 1.1+ T cells Blood, January 15, 2002; 99(2): 513 - 519. [Abstract] [Full Text] [PDF]
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T.-S. Migone, M. Humbert, A. Rascle, D. Sanden, A. D'Andrea, and J. A. Johnston The deubiquitinating enzyme DUB-2 prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal Blood, September 15, 2001; 98(6): 1935 - 1941. [Abstract] [Full Text] [PDF]
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J. J. Moon and B. H. Nelson Phosphatidylinositol 3-Kinase Potentiates, but Does Not Trigger, T Cell Proliferation Mediated by the IL-2 Receptor J. Immunol., September 1, 2001; 167(5): 2714 - 2723. [Abstract] [Full Text] [PDF]
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A. Martino, J. H. Holmes IV, J. D. Lord, J. J. Moon, and B. H. Nelson Stat5 and Sp1 Regulate Transcription of the Cyclin D2 Gene in Response to IL-2 J. Immunol., February 1, 2001; 166(3): 1723 - 1729. [Abstract] [Full Text] [PDF]
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Y.-J. Zhou, K. S. Magnuson, T. P. Cheng, M. Gadina, D. M. Frucht, J. Galon, F. Candotti, R. L. Geahlen, P. S. Changelian, and J. J. O'Shea Hierarchy of Protein Tyrosine Kinases in Interleukin-2 (IL-2) Signaling: Activation of Syk Depends on Jak3; However, Neither Syk nor Lck Is Required for IL-2-Mediated STAT Activation Mol. Cell. Biol., June 15, 2000; 20(12): 4371 - 4380. [Abstract] [Full Text]
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 C. Oetzel, T. Jonuleit, A. Götz, H. van der Kuip, H. Michels, J. Duyster, M. Hallek, and W. E. Aulitzky The Tyrosine Kinase Inhibitor CGP 57148 (ST1 571) Induces Apoptosis in BCR-ABL-positive Cells by Down-Regulating BCL-X Clin. Cancer Res., May 1, 2000; 6(5): 1958 - 1968. [Abstract] [Full Text]
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J. D. Lord, B. C. McIntosh, P. D. Greenberg, and B. H. Nelson The IL-2 Receptor Promotes Lymphocyte Proliferation and Induction of the c-myc, bcl-2, and bcl-x Genes Through the trans-Activation Domain of Stat5 J. Immunol., March 1, 2000; 164(5): 2533 - 2541. [Abstract] [Full Text] [PDF]
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D. E. Isaksen, H. Baumann, P. A. Trobridge, A. G. Farr, S. D. Levin, and S. F. Ziegler Requirement for Stat5 in Thymic Stromal Lymphopoietin-Mediated Signal Transduction J. Immunol., December 1, 1999; 163(11): 5971 - 5977. [Abstract] [Full Text] [PDF]
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A. E. Hunt, F. V. Lali, J. D. Lord, B. H. Nelson, T. Miyazaki, K. J. Tracey, and B. M. J. Foxwell Role of Interleukin (IL)-2 Receptor beta -Chain Subdomains and Shc in p38 Mitogen-activated Protein (MAP) Kinase and p54 MAP Kinase (Stress-activated Protein Kinase/c-Jun N-terminal Kinase) Activation. IL-2-DRIVEN PROLIFERATION IS INDEPENDENT OF p38 AND p54 MAP KINASE ACTIVATION J. Biol. Chem., March 12, 1999; 274(11): 7591 - 7597. [Abstract] [Full Text] [PDF]
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L. E. Cheng, C. Ohlen, B. H. Nelson, and P. D. Greenberg Enhanced signaling through the IL-2 receptor in CD8+ T cells regulated by antigen recognition results in preferential proliferation and expansion of responding CD8+ T cells rather than promotion of cell death PNAS, March 5, 2002; 99(5): 3001 - 3006. [Abstract] [Full Text] [PDF]
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